We have previously reported the presence of IGF-I and IGF-binding proteins (IGFBP-2, -3 and -4) in rat milk. Herein, the potential sources of rat milk IGF-I and IGFBPs were investigated. Lactating dams (day 14 postpartum) were separated from their pups and injected intraperitoneally with 0.45 microCi 125I-IGF-I or 125I-IGFBP-3. After 3 h, serum and milk of rats receiving 125I-IGF-I contained 7642 +/- 3121 and 14,455 +/- 7837 c.p.m./ml respectively. Serum and milk of rats given 125I-IGFBP-3 contained 7232 +/- 1366 and 10,371 +/- 4091 c.p.m./ml respectively. Sephacryl S-200 gel filtration chromatography demonstrated that the 125I-IGF-I in both serum and milk was primarily in the 150 kDa IGF-binding complex, whereas the distribution of 125I-IGFBP-3 differed between serum and milk. In serum, most of the 125I-IGFBP-3 was in the 150 kDa fraction, while most 125I-IGFBP-3 in milk was in the 40 kDa fraction. Northern analysis of liver showed IGFBP-1 and -3 mRNA expression, with variable expression of IGFBP-2 and -4 mRNA. In contrast, mammary tissue expressed only IGFBP-2 and -4 mRNA, suggesting that these IGFBPs in milk may arise from de novo synthesis within the mammary gland. The lack of detectable IGFBP-3 mRNA in mammary tissue and the translocation of 125I-IGFBP-3 from the serum suggest that milk IGFBP-3 arises from the maternal circulation.